Human pathogenic dermatophytes, which belong to the three genera Trichophyton, Microsporum and Epidermophyton, are fungi that infect human skin, nails, and hair. While the genus Epidermophyton is represented only by a single species (E. floccosum), the genera Microsporum and Trichophyton include several different species (1). Depending on the site of the infection the dermatophytosis can be divided in: Tinea barbae, an infection of the bearded area mainly caused by T. verrucosum and T. mentagrophytes; Tinea capitis, usually caused by organisms from genera Microsporum and Trichophyton; Tinea corporis by any of the human pathogenic dermatophytes; Tinea favosa by T. schoenleninii; Tinea pedis frequently caused by T. mentagrophytes, E. floccosum and T. rubrum; Tinea mannum caused by T. rubrum and Tinea unguinum (also termed onychomycosis) mainly caused by T. rubrum and T. mentagrophytes (1). Prevalence rates of onychymycosis in European countries vary between 3 and 22% (2). Thus, in a study by Summerbell et al. including 2662 nails, the following infective agents were isolated: T. rubrum (>70%), T. mentagrophytes (20%), Candida albicans (5.5%), and Scopulariopsis brevicaulis or non-dermatophyte moulds (1.6%) (3). Tinea unguium is the form of dermatophytosis that is most refractory to treatment and also relapses of infection in clinically cured nails are common (4). Evans et al. (5) compared the efficacy of terbinafine and itraconazole treatment of onychomycosis and showed that mycological cure was achieved in 76% of patients receiving terbinafine but only in 38% receiving itraconazole. In a later study also by Evans et al. the percentage of patients with mycological failure after standard treatment was 20% (6).
Topical therapy is sufficient in most of cases of dermatophyte skin infection, but long term and often expensive systemic treatment is necessary in cases of tinea capitis, tinea barbae and onychomycosis. Various side effects are associated with the systemic antifungals, e.g. gastro-intestinal side effects occur in 3-5% of the patients treated orally with terbinafine and—although less frequent—bone marrow suppression and hepatic side effects occur, why the liver function should be evaluated at baseline and periodically during treatment (7). The diagnosis of onychomycosis should be confirmed before therapy is initiated partly because of the complications associated with the treatment but also because other medical conditions and trauma may cause nail changes that may resemble onychomycosis (8).
Furthermore, genus and in some cases even the species-specific diagnosis is necessary due to different susceptibility patterns and contagious potential of the various dermatophytes, thus e.g. Microsporum species are less susceptible than Trichophyton spp. to terbinafine and should be treated with griseofulvin, T. rubrum strains are more susceptible to some antifungal drugs than T. mentagrophytes and some dermatophytes may cause epidemic outbreaks in schools and institutions due to transfer from man to man while others may not as their primary host is an animal (10).
The current diagnosis of dermatophytes is based on microscopic identification of spores and hyphae in clinical specimens followed by in vitro culture and morphological identification of the fungus (1). Direct microscopic examination of skin and nail material is often sufficient for the preemptive diagnosis of a fungal infection, but it does not give specific species diagnosis. Furthermore, although rapid and cheap, this technique has a relatively low sensitivity and shows false negative results in up to 15% cases (2). Application of culture enables specific species identification in 10-15 days in approximately 95% of cases. However, for some slow growing or atypical isolates time to diagnosis is up to 3-4 weeks. Such cases are especially cost- and time-consuming and require specialist skills (2).
It is thus obvious that a simple, rapid and specific method for the diagnosis of dermatophyte infections is necessary. Introduction of a PCR based methodology would increase specificity, simplicity, speed and on the same time be inexpensive.
For studies on species identification and typing, PCR (3,4) PCR fingerprinting (5), random amplification of polymorphic DNA (RAPD) (6), PCR and restriction fragment length polymorphism (RFLP) (7), arbitrarily primed PCR (AP-PCR) (8) have been applied. The main targets have been the following genes or DNA fragments: rDNA region, DNA topoisomerases II genes (11,14). chitin synthase gene (18).
A simple and fast extraction method of the DNA directly from patient samples is necessary for routine application of a molecular based detection methodology of dermatophyte infections. In previously published studies on the use of PCR for identification and/or typing of dermatophyte cultures, typically two steps are involved—disruption of fungi cells and subsequent DNA purification. The disruption of the fungal cells has been performed by mechanical disruption of the cell (grinding, freezing-thawing repeated steps, bead-beating) (16) and/or by chemical lysis (9) of the cell wall: enzymatic (proteinase K, zymolase) (12) or detergent lysis (10,15). The purification of the DNA from disrupted cells has been performed by application of phenol-chlorophorm (12,15) extraction method, by precipitation of DNA (10), or by using DNA's affinity to some specific resins (11) (commercial kits for purification of the DNA).
Such methodologies are inconvenient for routine diagnostic purposes for several reasons. First, the necessary initial cultivation of the patient samples is time consuming (up to 10 days) (13). Next, the phenol/chlorophorm DNA extraction method applied in the methods of the DNA extraction directly from skin and hair samples (14) is toxic and thus not applicable for routine diagnostic use in laboratories receiving a large number of samples per day. Finally, the previously published methods all involve a number of handlings (e.g. grinding or bead beating, and washing of the DNA pellets or columns) that increase the risk of the contamination of the samples and false PCR results. In agreement with this, PCR based diagnostic methods are not applied at a routine basis at any diagnostic laboratory to our knowledge, neither in Denmark nor worldwide.
The present invention solves this problem by disclosing a PCR based diagnosis of dermatophyte infections after a simple DNA extraction method which can be applied directly on clinical specimens.